Human embryonic stem cell-derived keratinocytes exhibit an epidermal transcription program and undergo epithelial morphogenesis in engineered tissue constructs

摘要

Human embryonic stem (hES) cells are an attractive source of cellular material for scientific, diagnostic, andpotential therapeutic applications. Protocols are now available to direct hES cell differentiation to specific lineagesat high purity under relatively defined conditions; however, researchers must establish the functionalsimilarity of hES cell derivatives and associated primary cell types to validate their utility. Using retinoic acid toinitiate differentiation, we generated high-purity populations of keratin 14þ (K14) hES cell-derived keratinocyte(hEK) progenitors and performed microarray analysis to compare the global transcriptional program of hEKsand primary foreskin keratinocytes. Transcriptional patterns were largely similar, though gene ontology analysisidentified that genes associated with signal transduction and extracellular matrix were upregulated in hEKs. Inaddition, we evaluated the ability of hEKs to detect and respond to environmental stimuli such as Ca2þ, serum,and culture at the air–liquid interface. When cultivated on dermal constructs formed with collagen gels andhuman dermal fibroblasts, hEKs survived and proliferated for 3 weeks in engineered tissue constructs. Maintenanceat the air–liquid interface induced stratification of surface epithelium, and immunohistochemistry resultsindicated that markers of differentiation (e.g., keratin 10, involucrin, and filaggrin) were localized tosuprabasal layers. Although the overall tissue morphology was significantly different compared with humanskin samples, organotypic cultures generated with hEKs and primary foreskin keratinocytes were quite similar,suggesting these cell types respond to this microenvironment in a similar manner. These results represent animportant step in characterizing the functional similarity of hEKs to primary epithelia.